1. Field of the Invention
The present invention relates to the methods of treatment of patients with heart disorders and more particularly to the method of using Fructose-1,6-Diphospate in treatment of the above mentioned diseases, and also as a protective agent against unforeseen catastrophic hypotension or hypoxia during operative procedures, and as a preservative agent for transplantation organs.
2. General Background and Prior Art
In medicine and physiology it is well-known that a continuous supply of energy is necessary for the function and maintenance of a living state by cells. The degree of intracellular energy is measured by the ratio of high energy phosphate compounds to those of less energy potential (i.e., adenosine triphosphate to adenosine diphosphate and adenosine monophosphate). The biochemical pathways which produce high energy phosphate compounds have been well established in the scientific literature as a chain of reactions that result in the breakdown of the major substrates, glucose or other sugars to pyruvic and lactic acid and is a process of carbohydrate metabolism. Although one stage of glycolysis requires oxidation by dehydrogenation, this may be accomplished without oxygen, so the process as a whole may be anaerobic. The pyruvic acid formed by glycolysis is then oxidized to carbon dioxide and water. This oxidation is the source of most of the utilizable energy (ATP) derived from carbohydrate metabolism. Glycolysis also yields some energy in the form of ATP which can be utilized for muscle contraction and other functions. This is particularly important during sudden strenuous exercise, when energy must be made available in excess of that which can be provided by oxidation processes.
The glycolytic process taking place in animal tissues involves the sequence of intermediates: ##STR1##
Fructose-1,6-diP is cleared by the enzyme aldolase between and third and fourth carbon atoms to form two triose phosphate molecules, glyceraldehyde-3-P and dihydroxyacetone phosphate. ##STR2##
This reaction is reversible. Glyceraldehyde-3-P and dihydroxyacetone-P are freely interconvertible through the action of triose-P-isomerase.
The next step in the main stream of glycolysis consists of the combined phosphorylation and oxidation of glyceraldehyde-3-P to 1,3 diphosphoglyceric acid, which is catalyzed by the enzyme glyceraldehyde-3-P dehydrogenase: EQU Glyceraldehyde-3-P+Pi+DPN+.revreaction.1,3Diphosphoglyceric acid+DPN.multidot.N+H.sup.+
The conversion of glyceraldehyde-3-P to 1,3 diphosphoglyceric acid proceeds anaerobically through oxidation by DPN.sup.+. In this process DPN.sup.+ is converted to DPN.sup..multidot. H, and the reaction would soon cease without a mechanism to reoxide DPN.sup..multidot. H to DPN.sup.+, since the amount of coenzyme present is very small. Anerobically DPN.sup..multidot. H is oxidized to DPN.sup.+ by pyruvic acid, with the formation of lactic acid: ##STR3##
Substances other than pyruvic acid may serve also to oxidize DPN.sup..multidot. H to DPN.sup.+. Among these are dihydroxyacetone-P, which is reduced to .alpha.-glycerophosphate, and oxaloacetic acid, which is reduced to malic acid. Reduction by these substances is of importance in starting the glycolytic process before sufficient pyruvic acid has been formed to function in the regeneration of DPN.sup.+. No ATP is formed in the oxidation of DPN.sup..multidot. H by pyruvic acid.
When the supply of oxygen to the tissues and the oxidative mechanisms are adequate, the DPN.sup..multidot. H is oxidized to DPN.sup.+ through the mitochondrial electron transport chain: EQU DPN.sup..multidot. H-FP-Cytochromes-O.sub.2 .fwdarw.DPN.sup.+ +H.sub.2 O+3ATP
Consequently, lactic acid accumulates in tissues only when oxidation by O.sub.2 cannot keep up with glycolytic reactions and pyruvic acid is reduced to lactic acid.
In this stage of glycolysis we have the first generation of utilizable energy as ATP. 1 molecule of ATP per triose phosphate mol when DPN.sup..multidot. H is oxidized anaerobically (by pyruvate), and 4 mols per triose phosphate mol when DPN.sup..multidot. H is oxidized by O.sub.2.
The next stage of glycolysis consists in the conversion of 3-phosphoglyceric acid to 2-phosphoglyceric acid by the enzyme phosphoglyceromutase, which requires catalytic amounts of 2,3-diphosphoglyceric acid.
Glycolysis proceeds by the conversion of 2-phosphoglyceric acid to phosphoenolpyruvic acid through action of the enzyme enolase. This reaction involves dehydration of 2-phosphoglyceric acid and is freely reversible. The loss of water converts the low-energy phosphate group of 2-phosphoglyceric acid to the high energy phosphate group of phosphopyruvic acid. Because of the high energy phosphate group present in phosphopyruvic acid, it reacts with ADP to form ATP and pyruvic acid to complete glycolysis properly. The reaction is catalyzed by the enzyme ATP-phosphopyruvic transphosphorylase or pyruvic kinase, which requires Mg.sup.++ and K.sup.+ for activation. This reaction accounts for the formation of 2 mols of ATP per mol of sugar glycolyzed.
The reactions of glycolysis in animal tissues lead to the end products pyruvic and lactic acids. Pyruvic acid is oxidized and converted to acetyl CoA by an oxidative .alpha.-ketodecarboxylase enzyme. Oxidation of the DPN.sup..multidot. H in the electron transport chain yields 3ATP per mol. The acetyl CoA formed from pyruvic acid is oxidized in the citric acid cycle to CO.sub.2 and H.sub.2 O with the formation of 12ATP per mol. So the aerobic pathway for the breakdown of sugars and fatty acids is dependent upon a ready supply of oxygen, which is provided to the body tissues through the bloodstream and is bound weakly to hemoglobin. Thus, interruption of either the pumping action of the heart occlusion of one of the arteries or failure to oxygenate the blood being circulated by the lungs will result in either a regional or generalized unavailability of oxygen.
If oxygen is not supplied to living tissue for any of the above mentioned reasons, aerobic or oxygen dependent metabolism ceases. This leads to an attempt to compensate the failure in oxygen supply by an increase in the rate of anaerobic metabolism.
It was already noted that the anaerobic metabolic pathway for carbohydrates involves glucose which then becomes phosphorated (has six-carbon sugar). These molecules then break down to trioses (three-carbon sugars) and enter the aerobic pathway as the pyruvate molecule. In tissues that have limited blood supply or, for some other reason fail to be given an adequate amount of oxygen, the aerobic pathway must provide all of the energy necessary for cellular function. However, during any form of oxygen deprivation, whether it is from heart attack or from blood loss, leading to hypoperfusion as in an injury, the pathways of metabolism are refractory to the further entrance of the glucose into the cell, and the critical breakdown point in the metabolic pathway is at the phospho-fructo-kinase enzyme step. This means that unless something different is done, the individual deteriorates to the point where his tissues are unable, because of the injury, to regain function which may lead to a fatal outcome.
Investigations have been made on the effect of sugars on the recovery of heart functions. For example, Dr. Pasi Kettunen of Finland described his experiments on the "Comparison of the Effect of Glucose and Fructose on the Recovery of the Heart Preparation" (Scand. j. clin. Lab. Invest. 37, 705-708, 1977). Potassium citrate solution was used for heart arrest, and heart function was recovered by infusion of Locke's solution, plus glucose, fructose or sucrose. During recovery period, the amplitude and frequency of heart beats, the lactic acid in the drained perfusion solution, pH and potassium concentration were measured. The use of glucose, fructose or sucrose made no significant difference to any of these parameters. Next, the metabolism of glucose and fructose in the heart was investigated and on a metabolic basis the use of glucose for resuscitation would seem to be more appropriate than fructose.
Dr. L. H. OPie and P. Owen in their investigation of glycolysis in acute experimental myocardial infarction found out that glycolysis during anaerobic circumstances may be accelerated by all the factors that stimulate phosphofructokinase activity. They indicated that there is an overwhelming change that may be expected to inhibit phosphofructokinase activity, namely the intracellular accumulation of hydrogen ions. This phenomena was confirmed by Ui in 1960 and Kubler and Spieckermann in 1970. Dr. Opie then wrote that some index of phosphofructokinase activity can be obtained by measurements of tissue contents of glucose-6-diphosphate. Glycolytic flow may be compared to a regular stream, phosphofructokinase acts as a am-wall, inhibiting the flow of glycolysis. Thus it is evident that these investigations, being valuable by themselves could not provide a sufficient method and an agent which can modify energy requirements intracellularly in the fact of low oxygen levels, poor blood circulation or poor distribution of circulation.